concanavalin-a and methylglucoside

A Systematic investigation of the effect of pH on concanavalin A in the presence of specific and non-specific sugars is made using CD (circular dichroism) and fluorescence. The specific and non-specific sugars for concanavalin A were methyl alpha-D-glucopyranoside and methyl alpha-D-galactopyranoside respectively. Far-UV CD showed changes in the MRE value at 217 nm in the presence of the above-mentioned sugars. At pH 7, the CD and fluorescence spectra obtained in the presence of methyl alpha-D-glucopyranoside were slightly different from those for the native state and a significant difference was obtained in the presence of methyl alpha-D-galactopyranoside. Near-UV CD spectra showed the retention of a native-like tertiary structure in the presence of the specific sugar upon pH denaturation. Tryptophan fluorescence studies indicated a change in the tryptophan enviornment. The results obtained from our CD data are consistent with those obtained from fluorescence studies. Upon pH exposure of concanavalin A in the presence of methyl alpha-D-glucopyranoside and methyl alpha-D-galactopyranoside, the former acted as a protector preventing conformational alteration at different pH while the presence of latter induced a different stable conformational state and this state persists over the pH range from 2 to 10.

Topics: Circular Dichroism; Concanavalin A; Galactose; Hydrogen-Ion Concentration; Methylglucosides; Spectrometry, Fluorescence

Layered thin films composed of concanavalin A (Con A) and sugar-bearing polymers were prepared by a layer-by-layer deposition of Con A and the polymer on a solid surface. The sugar-induced disintegration was studied. Con A-polymer layered films could be successfully prepared using a maltose-bearing polymer (PV-MA), while melibiose- and glucose-bearing polymers (PV-MEA and PV-G) did not afford a layered film, due to a weak affinity of PV-MEA and PV-G to Con A. The Con A/PV-MA layered film was stable in pH 7 and 8 solutions, while in a pH 6 medium the film was slightly unstable. The Con A/PV-MA film was disintegrated upon the addition of sugars in solution owing to a preferential binding of the sugars to the binding site of Con A in the film. The disintegration rate was dependent on the type of sugar and its concentration. The Con A/PV-MA film was disintegrated rapidly upon the addition of methyl alpha-D-mannopyranoside, while the rate was slower upon the addition of the same concentration of D-mannose, D-glucose and methyl alpha-D-glucopyranoside. The present system may be useful for constructing sensitive devices that can release a drug or other functional molecules in response to sugars.

Topics: Carbohydrates; Concanavalin A; Galactose; Glucose; Maltose; Mannose; Melibiose; Methylglucosides; Methylmannosides; Polyvinyls; Spectrophotometry, Ultraviolet

Lactobacillus gasseri is a common inhabitant of human intestine. The L. gasseri strains SBT10239 and SBT10241 have shown high antimutagenicity and binding properties with different heterocyclic amines. In order to identify the cell wall components involved in binding with the heterocyclic amines, the cells and cell walls of L. gasseri strains were subjected to different chemical and enzymatical treatments, prior to the binding experiments. The results indicated that the binding receptors for heterocyclic amines are the carbohydrate moieties of the cell wall. Binding of the heterocyclic amines with L. gasseri cell walls and the carbohydrate content showed high correlation coefficient, whereas it was insignificant or negative with protein content. The lectin binding studies revealed that the glucose molecules of the cell wall has a significant role in binding the heterocyclic amines. The inhibition caused by the lectin Concanavalin A was reversed when treated with methyl glucoside, a competitive inhibitor of Concanavalin A and restored the binding of heterocyclic amine with the cells.

Topics: Antimutagenic Agents; Binding, Competitive; Carbohydrates; Carbolines; Cell Wall; Concanavalin A; Humans; Lactobacillus; Methylglucosides; Mutagens

Wheat germ agglutinin binding to a rat hepatoma cell line dRLa 74 treated with concanavalin A was studied. It increased depending on the concanavalin A concentration in the culture medium. The cells exhibited about twofold increase in wheat germ agglutinin-binding when pretreated with 50 micrograms/ml of concanavalin A for 48 h. The wheat germ agglutinin binding sites were shown to be localized at the cell surface by lectin-histochemistry. Wheat germ agglutinin blotting of microsomal membrane proteins showed a broad wheat germ agglutinin-reactive band with an apparent molecular weight of 90 to 100 kDa. Loss of wheat germ agglutinin binding to dRLa 74 cells and the glycoprotein after neuraminidase treatment suggested that wheat germ agglutinin reacted with cell surface sialyl residues of dRLa 74 cells. The induced change was reversible. Increased wheat germ agglutinin binding returned to the pretreatment level when the concanavalin A-treated cells were subcultured in the absence of concanavalin A. These observations suggest that environmental factors interacting with tumor cell surface sugar moieties may induce reversible epigenetic changes on cell surface carbohydrate structures.

Topics: Animals; Concanavalin A; Electrophoresis, Polyacrylamide Gel; Histocytochemistry; Immunoblotting; Liver Neoplasms, Experimental; Methylglucosides; Ovalbumin; Rats; Receptors, Mitogen; Tumor Cells, Cultured; Wheat Germ Agglutinins

Extracellular calcium (Ca2+) is the major physiological regulator of parathyroid function; high Ca2+ decreases PTH secretion as well as reduces cAMP accumulation. There is an increasing body of evidence suggesting the presence of a receptor-like mechanism at the surface of the parathyroid cell which mediates these and other actions of Ca2+. In the present studies we used the lectin Concanavalin-A (Con-A) to investigate the possible role of carbohydrate moieties in the regulation of cAMP metabolism by Ca2+ in bovine parathyroid cells, which is thought to involve inhibition of adenylate cyclase via activation of the guanine nucleotide regulatory protein Gi. Pretreatment of parathyroid cells with Con-A for 15-60 min significantly reversed the inhibitory effect of high Ca2+ on dopamine-stimulated cAMP accumulation, reducing the inhibition at 3 mM Ca2+ from 70 +/- 3% to 30 +/- 3%. This effect was also observed in the absence of preincubation and with concentrations of Con-A as low as 40 micrograms/ml and was reversed by alpha-methyl-D-glucoside, a specific antagonist of the lectin. The lectin also reversed the inhibitory effects of Ca2+ (2-3 mM) on cAMP accumulation stimulated by isoproterenol and forskolin to a comparable extent. Prostaglandin F2 alpha-induced inhibition of cAMP accumulation (likewise mediated by Gi) was, however, not reversed by Con-A, suggesting that the lectin did not have a generalized effect on the cell surface or on receptors inhibiting adenylate cyclase. Moreover, fluoride-induced inhibition of cAMP accumulation was not reversed by Con-A, providing additional evidence that the lectin did not act at or distal to Gi (i.e. modulate Gi, adenylate cyclase, and/or phosphodiesterase). The present study suggests that Con-A may modulate the actions of extracellular Ca2+ on parathyroid secretion, possibly modifying the interaction of Ca2+ with the cell surface by affecting carbohydrate moieties that seem to be important in the Ca2(+)-sensing process. The structural element involved in Ca2+ sensing in the parathyroid cell may be a glycoprotein or closely associated with glycoproteins with carbohydrate chains containing alpha-methyl-D-glycoside.

Topics: Animals; Calcium; Cattle; Colforsin; Concanavalin A; Cyclic AMP; Dinoprost; Dopamine; Fluorides; Ions; Isoproterenol; Methylglucosides; Osmolar Concentration; Parathyroid Glands

We report here the preparation of iron-containing concanavalin A. It has a protein-to-iron ratio of 2.0, and the iron compound it contains is particulate with an average diameter of 85 A. Iron-containing concanavalin A interacts reversibly with dextran and with methyl alpha-D-glucoside. The molecular basis of these findings is discussed and a possible mechanism suggested where one of the molecular forms of concanavalin A has the structure of an apoferritin into which iron is deposited in the form of ferrihydrite.

Topics: Chemical Phenomena; Chemical Precipitation; Chemistry; Concanavalin A; Dextrans; Ferritins; Ferrous Compounds; Iron; Macromolecular Substances; Methylglucosides; Molecular Weight; Protein Conformation; Quaternary Ammonium Compounds; Spectrometry, Gamma

Succinylated concanavalin A (SCon A) lyses sheep erythrocytes (E) in the presence of complement, whereas the native tetravalent lectin, Con A, is inactive. We have studied the ability of E-SCon A (ES) to interact with early acting guinea pig (gp) or human (hu) complement components (C1, C2, C4) and found that cell intermediates ESC1, ESC4, ESC14, and ESC142 can be generated that are analogous to intermediates conventionally prepared with E and rabbit IgM (pentameric) anti-Forssman antibody. Titration of gp or hu C1, C4, and C2, and quantification of the number of activated C1 molecules bound to ESC4 by the C1 fixation and transfer test showed in each case that an average of one effective site per cell was sufficient to cause cell lysis. Determination of tmax for optimal formation of ESC142 sites depended on the species combination of components used to make the intermediates, and the decay of ESC142 and EAC142 sites or sites generated with ESC4, EAC4, and trypsin-activated C2 were similar. The sugar alpha-D-methylglucopyranoside (alpha-MGP) inhibited binding of SCon A to E and eluted the lectin from ES, whereas galactose was nearly inactive, consistent with lectin sugar-binding selectivity. In contrast, both sugars were ineffective in eluting SCon A or C4hu from ESC4hu, indicating that C4hu blocked the interaction between lectin and alpha-MGP, perhaps by steric hindrance. SCon A is a divalent functional analogue of IgM anti-Forssman antibody that may be a uniquely suited reagent specific for cell membrane glycoconjugates for studying the mechanism of binding and activation of complement components.

Topics: Animals; Binding Sites, Antibody; Complement Activation; Complement C1; Complement C2; Complement C4; Complement Fixation Tests; Concanavalin A; Erythrocytes; Guinea Pigs; Hemolysis; Humans; In Vitro Techniques; Methylglucosides; Rats

The probable modes of binding of Methyl--alpha (and beta)-D-glucopyranosides and some of their derivatives to concanavalin A have been proposed from theoretical studies. Theory predicts that beta-MeGlcP can bind to ConA in three different modes whereas alpha-MeGlcP can bind only in one mode. beta-MeGlcP in its most favourable mode of binding differs from alpha-MeGlcP in its alignment in the active-site of the lectin where it binds in a flipped or inverted orientation. Methyl substitution at the C-2 atom of the alpha-MeGlcP does not significantly affect the possible orientations of the sugar in the active-site of the lectin. Methyl substitution at C-3 or C-4, however, affects the allowed orientations drastically leading to the poor inhibiting power of Methyl-3-O-methyl-alpha-D-glucopyranoside and the inactivity of Methyl-4-O-methyl-alpha-D-glycopyranoside. These studies suggest that the increased activity of the alpha-MeGlcP over beta-MeGlcP may be due to the possibility of formation of better hydrogen bonds and to hydrophobic interactions rather than to steric factors as suggested by earlier workers. These models explain the available NMR and other binding studies.

Topics: Binding Sites; Carbohydrate Conformation; Concanavalin A; Hydrogen Bonding; Methylglucosides; Methylglycosides; Models, Chemical; Stereoisomerism

We purified the urinary procoagulant from frozen human urine by introducing phenyl-Sepharose hydrophobic chromatography. By this method, the apoprotein of the procoagulant and the lipid-like substance were separately recovered. Upon reassociation with the lipid-like substance or exogenous crude phospholipids, the apoprotein accelerated factor VIIa-catalyzed activation of factor X, probably by forming a stoichiometric complex with the catalytic enzyme. Thus the procoagulant was confirmed to be a tissue factor by its mode of participation in the blood coagulation mechanism.

Topics: Apoproteins; Blood Coagulation; Concanavalin A; Factor VII; Factor VIIa; Factor X; Humans; In Vitro Techniques; Male; Methylglucosides; Phospholipids; Thromboplastin

Topics: Antigens; Blood Protein Electrophoresis; Concanavalin A; Factor VIII; Humans; Methylglucosides; von Willebrand Diseases; von Willebrand Factor

Microsomal and lysosomal beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase, EC 3.2.1.31) of monkey brain were differentially eluted from Con A-Sepharose when subjected to chromatography and linear gradient elution with methyl alpha-glucoside at 28+/-1 degree C. The lysosomal enzyme was eluted as a sharp peak in the first few fractions, while the microsomal enzyme was eluted as a broad peak extending over several fractions. This differential pattern of elution was dependent only on the temperature of elution and the concentration of methyl alpha-glucoside used. The lysosomal and microsomal glucuronidases were purified to apparent homogeneity and their neutral sugar analysed. Both of them contained glucose, mannose and fucose but the microsomal enzyme contained about 3-times as much of all these sugars as the lysosomal enzyme. Sodium periodate treatment of the microsomal enzyme resulted in a shift in its elution pattern, similar to the lysosomal enzyme when subjected to Con A-Sepharose chromatography. The content of neutral sugars and the structural features of the oligosaccharide units in the microsomal glucuronidase might be responsible for its elution pattern. A processing of the carbohydrate units of the microsomal glucuronidase might be envisaged to take place if it were to act as a precursor of the lysosomal glucuronidase.

Topics: Animals; Brain; Carbohydrates; Chromatography, Affinity; Concanavalin A; Glucuronidase; Lysosomes; Macaca radiata; Methylglucosides; Microsomes; Periodic Acid

Cells of a pig Fallopian tube line were agglutinated by concanavalin A (Con A). Similar ConA agglutination was shown in diploid and nontumoral transformed cells with one (T1) translocation until the 112th subculture. Then a progressive increase of agglutination was shown until the 149th subculture and the rate of agglutination remained constant thereafter. The progression of ConA agglutination paralleled the progression of anchorage independence. The highest ConA agglutination occurred when anchorage independence was well established and persisted until the 193rd T1 subculture when benign tumorigenicity was demonstrated. After the appearance of the second translocation (T2), the self-agglutination of the cells was directly related to malignancy and to high tumor incidence in mice. Anchorage independence and the highest ConA agglutination were directly related to benign transformation and indirectly related to malignant transformation of the pig Fallopian tube cell line.

Topics: Agglutination; Animals; Cell Adhesion; Cell Aggregation; Cell Line; Cell Transformation, Neoplastic; Concanavalin A; Fallopian Tubes; Female; Methylglucosides; Swine